Sudden cardiac death is the single most common cause of death in industrialized countries, accounting for more than half of deaths from cardiovascular diseases. Most sudden cardiac deaths are caused by ventricular tachyarrhythmias, and an important risk factor for such arrhythmias is medications. A number of non- cardiovascular drugs have been shown to alter cardiac electrophysiology, and in some cases, the occurrence of serious ventricular arrhythmias has led to their withdrawal from the market. The macrolide antibiotics erythromycin and clarithromycin have been linked to life-threatening arrhythmias, while azithromycin is considered to have minimal adverse cardiac effects. However, recent case reports of serious ventricular arrhythmias, along with data from the FDA's Adverse Events Reporting System, have challenged this assumption. We have performed a large pharmacoepidemiologic study using the Tennessee Medicaid database, demonstrating that azithromycin increases the risk of sudden cardiac death by several fold. Moreover, case reports indicate that while QT prolongation is rarely causative, polymorphic ventricular tachycardia can occur in the absence of ECG abnormalities, implying novel pharmacologic effects, a concept also supported by our preliminary data. The mechanism of this unusual proarrhythmic syndrome is currently not known, and the electrophysiologic effects of azithromycin remain understudied. The goal of this proposal is to test the hypothesis that azithromycin causes novel electrophysiologic effects in the heart that increase susceptibility to serious ventricular arrhythmias. In Specific Aim 1, we will obtain intracellular calcium fluorescence measurements in intact cardiomyocytes to test the hypothesis that azithromycin increases the risk of sudden cardiac death by promoting abnormal spontaneous calcium release from the sarcoplasmic reticulum. This mechanism has been linked to polymorphic ventricular tachycardia in the setting of a normal QT interval in inherited arrhythmia syndromes. In Specific Aim 2, we will perform electrophysiologic studies to test the hypothesis that azithromycin alters ionic currents other than hERG to prolong repolarization and increase arrhythmia susceptibility. Currents will be studied following heterologous expression of recombinant human channels, as well as in native ventricular myocytes. The studies outlined in this proposal will improve our understanding of proarrhythmic mechanisms in humans, and specifically for the widely-prescribed antibiotic azithromycin. This knowledge would enable the screening and identification of additional compounds in drug development with similar properties that could also have the potential for serious adverse effects. Thus, an improved understanding of the basic mechanisms causing azithromycin-induced sudden cardiac death should lead to safer pharmacotherapy.